Enzymatic textile bleaching compositions and methods of use thereof

ABSTRACT

Described are compositions and methods for enzymatic bleaching of textiles. A perhydrolase enzyme is used in combination with an ester substrate and hydrogen peroxide to produce a peracid for textile bleaching. Textiles bleached by the exhibit increased dye uptake, decreased textile damage, and/or bulkier softer handle than textiles bleached by conventional chemical bleaching processes.

PRIORITY

The present application is a divisional of U.S. patent application Ser.No. 13/063,140, filed Sep. 15, 2011, which is a U.S. National Stageapplication of International Application No. PCT/US2009/56499, filedSep. 10, 2009, which claims priority to U.S. Provisional PatentApplication Ser. Nos. 61/095,807, filed on Sep. 10, 2008, 61/099,020,filed on Sep. 22, 2008, and 61/156,593, filed on Mar. 2, 2009, each ofwhich is incorporated by reference in its entirety.

TECHNICAL FIELD

The compositions and methods relate to the enzymatic bleaching oftextiles.

BACKGROUND

In the processing of textile fibers, yarns and fabrics, a pretreatmentor preparation step is typically required to properly prepare thenatural materials for further use, in particular, for the dyeing,printing, and/or finishing stages typically required for commercialgoods. These textile treatment steps remove impurities and color bodieswhich exist either naturally or are added to the fibers and/or fabricsduring spinning or weaving.

Textile manufacturing typically includes a number of treatments andstages, the most common being de-sizing (i.e., the removal of sizingagents, such as starches, via enzymatic, alkali or oxidative soaking);scouring (i.e., the removal of greases, oils, waxes, pectic substances,motes, protein and fats by contact with a solution of sodium hydroxideat temperatures near boiling); and bleaching (i.e., the removal andlightening of color bodies from textiles by conventional using oxidizingagents, such as hydrogen peroxide, hypochlorite, and chlorine dioxide,or by using reducing agents, such as, sulfur dioxide or hydrosulfitesalts). Currently employed bleaching technology involves the use ofalkaline hydrogen peroxide bleaching at temperatures in excess of 95° C.Such high temperatures and strong bleaching systems require high energyinput and typically produce high pH effluent, which is undesirable fromthe perspective of environmental sustainability.

There is a need for an effective enzymatic textile bleaching processthat minimizes the environmental footprint and costs of textile millsand provides improved fabric strength retention and reduced fiber damagecompared to conventional textile bleaching processes. Such an enzymaticbleaching process would preferably operate at a lower pH and lowertemperature, decrease the use of caustic chemicals, and be moreenvironmentally friendly than conventional methods.

BRIEF SUMMARY

The present compositions and methods relate to enzymatic textilebleaching. Use of the textile bleaching compositions and methods producebleached textiles with decreased textile damage, bulkier softer handle,and/or increased dye uptake when compared to a chemical textilebleaching method.

In one aspect, an enzymatic textile bleaching composition is provided,comprising: (i) a perhydrolase enzyme; (ii) an ester substrate for saidperhydrolase enzyme; (iii) a hydrogen peroxide source; (iv) a surfactantand/or an emulsifier; (v) a peroxide stabilizer; (vi) a sequesteringagent; and (vii) a buffer that maintains a pH of about 6 to about 8.

In some embodiments, the perhydrolase enzyme comprises the amino acidsequence set forth in SEQ ID NO: 1 or a variant or homolog thereof. Inparticular embodiments, the perhydrolase enzyme is the S54V variant ofSEQ ID NO: 1 (i.e., a variant of SEQ ID NO: 1 having the substitutionS54V). In some embodiments, the perhydrolase enzyme comprises (i.e.,exhibits) a perhydrolysis to hydrolysis ratio greater than 1. In someembodiments, the perhydrolase enzyme is present at a concentration ofabout 1 to about 2.5 ppm, for example, about 1.7 ppm.

In some embodiments, the ester substrate is selected from propyleneglycol diacetate, ethylene glycol diacetate, triacetin, ethyl acetate,and tributyrin. In a particular embodiment, the ester substrate ispropylene glycol diacetate. In some embodiments, propylene glycoldiacetate is present in the composition in an amount of about 2,000 toabout 4,000 ppm, for example, about 3,000 ppm.

In some embodiments, the hydrogen peroxide source is hydrogen peroxide.In some embodiments, hydrogen peroxide is present at a concentration ofabout 1,000 to about 3,000 ppm, for example, about 2,100 ppm.

In some embodiments, the surfactant and/or emulsifier comprises anon-ionic surfactant. In one embodiment, the non-ionic surfactant is analcohol ethoxylate. In one embodiment, the surfactant and/or emulsifiercomprises an isotridecanol ethoxylate. In one embodiment, the surfactantand/or emulsifier comprises an alcohol ethoxylate and an isotridecanolethoxylate. In one embodiment, the composition comprises a surfactantand an emulsifier.

In some embodiments, the enzymatic textile bleaching compositioncomprises a peroxide stabilizer and/or a sequestering agent. In oneembodiment, the peroxide stabilizer is phosphonic acid. In oneembodiment, the sequestering agent is polyacrylic acid.

In some embodiments, the composition further comprises a bioscouringenzyme. In some embodiments, the bioscouring enzyme is selected frompectinases, cutinases, cellulases, hemicellulases, proteases, andlipases. In one embodiment, the bioscouring enzyme is a pectinase.

In another aspect, a method for bleaching a textile is provided,comprising contacting the textile with an enzymatic textile bleachingcomposition as described herein for a length of time and underconditions suitable to permit measurable whitening of the textile,thereby producing a bleached textile, wherein the bleached textilecomprises at least one of decreased textile damage, bulkier softerhandle, and increased dye uptake when compared to a chemical textilebleaching method that comprises contacting the textile with a chemicaltextile bleaching composition that does not comprise a perhydrolaseenzyme. In some embodiments, the method further comprises hydrolyzinghydrogen peroxide with a catalase enzyme after the bleached textile isproduced. In one embodiment, the liquor ratio is about 10:1. In someembodiments, the method is performed in a batch or exhaust process.

In some embodiments, the method provides any of at least about 10, 20,30, 40, or 50% less weight loss than a chemical bleaching compositionthat does not comprise a perhydrolase enzyme.

In some embodiments, the method provides a textile capable of increaseddye uptake to produce a dyed textile with at least about any of at leastabout 5, 10, 15, 20, 25, or 30% increased dye depth when compared to atextile treated with a chemical bleaching composition that does notcomprise a perhydrolase enzyme.

In some embodiments, the method provides a textile that demonstrated(i.e., exhibits or possesses) reduced pilling propensity when comparedto a textile treated with a chemical bleaching composition that does notcomprise a perhydrolase enzyme.

In some embodiments, the textile is contacted with the enzymatic textilebleaching composition at a bleaching temperature of about 60° to about70° C. for a processing time of about 40 to about 60 minutes. In someembodiments, the temperature of the enzymatic textile bleachingcomposition is raised by about 3° C. per minute from a startingtemperature of about 20° to about 40° C. until the bleaching temperatureis reached. In one embodiment, the bleaching temperature is about 65° C.and the processing time is about 50 minutes.

In some embodiments, the bleached textile is rinsed with an aqueouscomposition at a rinsing temperature of about 40° C. to about 60° C. toremove said enzymatic textile bleaching composition. In one embodiment,the rinsing temperature is about 50° C. In one embodiment, rinsingcomprises rinsing said bleached textile twice for about 10 minutes foreach rinse. In some embodiments, the aqueous composition comprises acatalase enzyme to hydrolyze the hydrogen peroxide.

In another aspect, use of an enzymatic textile bleaching composition forbleaching a cellulose-containing textile is provided, the compositioncomprising an enzymatic textile bleaching composition as describedherein, characterized in that treating the textile with the compositionprovides improved dye uptake, bulkier softer handle, and/or decreasedtextile damage as compared to treatment with chemical bleaching.

DETAILED DESCRIPTION

The present compositions and methods relate to the enzymatic bleachingof textiles using a perhydrolase enzyme. The described enzymaticprocesses result in textiles with a bulkier softer handle, increased dyeuptake, and/or decreased textile damage when compared with a chemicalbleaching process. The processes are generally performed at a lowertemperature and with a lower rinsing requirement than traditionalchemical bleaching processes, resulting in energy and water savings. Theeffluent from the enzymatic bleaching process also has a lower pH (i.e.,<8) than that of a conventional chemical bleaching process (i.e., about13), thereby reducing the environmental impact of textile bleaching.

Unless otherwise indicated, the practice of the present compositions andmethods will employ conventional techniques in the fields of molecularbiology (including recombinant techniques), microbiology, cell biology,and biochemistry, which are known to those skilled in the art. Suchtechniques are describe in the literature, for example, MolecularCloning: A Laboratory Manual, second edition (Sambrook et al., 1989);Oligonucleotide Synthesis (M. J. Gait, ed., 1984; Current Protocols inMolecular Biology (F. M. Ausubel et al., eds., 1994); PCR: ThePolymerase Chain Reaction (Mullis et al., eds., 1994); and Gene Transferand Expression: A Laboratory Manual (Kriegler, 1990).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Singleton, et al., Dictionary of Microbiology and MolecularBiology, second ed., John Wiley and Sons, New York (1994), and Hale &Markham, The Harper Collins Dictionary of Biology, Harper Perennial, NY(1991) provide a general dictionary for reference.

Unless otherwise specified, numeric ranges are inclusive of the numbersdefining the range, nucleic acids are written left to right in 5′ to 3′orientation, and amino acid sequences are written left to right in aminoto carboxy orientation. Unless context clearly dictates otherwise, thearticles “a,” “an,” and “the” include both singular and pluralreferents. Unless otherwise specified, any methods and materials similaror equivalent to those described can be used in the practice or testingof the present compositions and methods. All reference cited herein arehereby incorporated by reference.

DEFINITIONS

The following terms and phrases are defined for clarity:

As used herein, the term “bleaching” refers the process of treating atextile material such as a fiber, yarn, fabric, garment or non-wovenmaterial to produce a lighter color. Bleaching encompasses the whiteningof a textile by removal, modification, or masking of color-causingcompounds in cellulosic or other textile materials. Thus, “bleaching”refers to the treatment of a textile for a sufficient length of time andunder appropriate pH and temperature conditions to effect a brightening(i.e., whitening) of the textile. Bleaching may be performed usingchemical bleaching agent(s) and/or enzymatically generated bleachingagent(s). Examples of suitable bleaching agents include but are notlimited to ClO₂, H₂O₂, peracids, NO₂, and the like.

As used herein, the term “bleaching agent” encompasses anymoiety/chemical that is capable of bleaching a textile. A bleachingagent may require the presence of a bleach activator. Examples ofsuitable chemical bleaching agents are sodium peroxide, sodiumperborate, potassium permanganate, and peracids. H₂O₂ may be considereda chemical bleaching agent when it has been generated enzymatically insitu. A “chemical bleaching composition” contains one or more chemicalbleaching agent(s).

As used herein, an enzyme is a protein (polypeptide) having catalyticactivity.

As used herein, an “enzymatic bleaching system” or “enzymatic bleachingcomposition” includes one or more enzyme(s) and substrate(s) capable ofenzymatically generating a bleaching agent. For example, an enzymaticbleaching system may contain a perhydrolase enzyme, an ester substrate,and a hydrogen peroxide source, for production of a peracid bleachingagent.

As used herein, an “ester substrate,” with reference to an enzymaticbleaching system containing a perhydrolase enzyme, refers to aperhydrolase substrate that contains an ester linkage. Esters comprisingaliphatic and/or aromatic carboxylic acids and alcohols may be utilizedas substrates with perhydrolase enzymes. In some embodiments, the estersource is an acetate ester. In some embodiments, the ester source isselected from one or more of propylene glycol diacetate, ethylene glycoldiacetate, triacetin, ethyl acetate and tributyrin. In some embodiments,the ester source is selected from the esters of one or more of thefollowing acids: formic acid, acetic acid, propionic acid, butyric acid,valeric acid, caproic acid, caprylic acid, nonanoic acid, decanoic acid,dodecanoic acid, myristic acid, palmitic acid, stearic acid, and oleicacid.

As used herein, the term “perhydrolase” refers to an enzyme that iscapable of catalyzing a perhydrolysis reaction that results in theproduction of a sufficiently high amount of peracid suitable for use ina textile bleaching method as described. Generally, a perhydrolaseenzyme exhibits a high perhydrolysis to hydrolysis ratio. In someembodiments, the perhydrolase comprises, consists of, or consistsessentially of the Mycobacterium smegmatis perhydrolase amino acidsequence set forth in SEQ ID NO: 1, or a variant or homolog thereof. Insome embodiments, the perhydrolase enzyme comprises acyl transferaseactivity and catalyzes an aqueous acyl transfer reaction.

As used herein, a “peracid” is an organic acid of the formula RC(═O)OOH.

As used herein, the term “hydrogen peroxide source” refers to hydrogenperoxide that is added to a textile treatment bath either from anexogenous (i.e., an external or outside) source or generated in situ bythe action of a hydrogen peroxide generating oxidase on a substrate. A“hydrogen peroxide source” includes hydrogen peroxide as well as thecomponents of a system that can spontaneously or enzymatically producehydrogen peroxide as a reaction product.

The phrase “perhydrolysis to hydrolysis ratio” refers to the ratio ofthe amount of enzymatically produced peracid to the amount ofenzymatically produced acid by a perhydrolase enzyme from an estersubstrate under defined conditions and within a defined time. In someembodiments, the assays provided in WO 05/056782 are used to determinethe amounts of peracid and acid produced by the enzyme.

As used herein, the term “acyl” refers to an organic group with thegeneral formula RCO—, which can be derived from an organic acid byremoval of the —OH group. Typically, acyl group names end with thesuffix “-oyl,” e.g., methanoyl chloride, CH₃CO—Cl, is the acyl chlorideformed from methanoic acid, CH₃CO—OH).

As used herein, the term “acylation” refers to a chemical transformationin which one of the substituents of a molecule is substituted by an acylgroup, or the process of introduction of an acyl group into a molecule.

As used herein, the term “transferase” refers to an enzyme thatcatalyzes the transfer of a functional group from one substrate toanother substrate. For example, an acyl transferase may transfer an acylgroup from an ester substrate to a hydrogen peroxide substrate to form aperacid.

As used herein, the term “hydrogen peroxide generating oxidase” means anenzyme that catalyzes an oxidation/reduction reaction involvingmolecular oxygen (O₂) as the electron acceptor. In such a reaction,oxygen is reduced to water (H₂O) or hydrogen peroxide (H₂O₂). An oxidasesuitable for use herein is an oxidase that generates hydrogen peroxide(as opposed to water) on its substrate. An example of a hydrogenperoxide generating oxidase and its substrate suitable for use herein isglucose oxidase and glucose. Other oxidase enzymes that may be used forgeneration of hydrogen peroxide include alcohol oxidase, ethylene glycoloxidase, glycerol oxidase, amino acid oxidase, and the like. In someembodiments, the hydrogen peroxide generating oxidase is a carbohydrateoxidase.

As used herein, the term “textile” refers to fibers, yarns, fabrics,garments, and non-wovens. The term encompasses textiles made fromnatural, synthetic (e.g., manufactured), and various natural andsynthetic blends. Thus, the term “textile(s)” refers to unprocessed andprocessed fibers, yarns, woven or knit fabrics, non-wovens, andgarments. In some embodiments, a textile contains cellulose.

As used herein, the phrase “textile(s) in need of processing” refers totextiles that need to be desized and/or scoured and/or bleached or maybe in need of other treatments such as biopolishing.

As used herein, the phrase “textile(s) in need of bleaching” refers totextiles that need to be bleached without reference to other possibletreatments. These textiles may or may not have been already subjected toother treatments. Similarly, these textiles may or may not needsubsequent treatments.

As used herein, the term “fabric” refers to a manufactured assembly offibers and/or yarns that has substantial surface area in relation to itsthickness and sufficient cohesion to give the assembly useful mechanicalstrength.

As used herein, the phrase “effective amount of perhydrolase enzyme”refers to the quantity of perhydrolase enzyme necessary toachieve/produce the enzymatic activity required in the subject processesor methods. Such effective amounts are readily ascertained by one ofordinary skill in the art, and are based on many factors, such as theparticular enzyme variant used, the pH used, the temperature used andthe like, as well as the results desired (e.g., level of whiteness).

As used herein, the term “oxidizing chemical” refers to a chemical thathas the ability to bleach a textile. The oxidizing chemical is presentat an amount, pH, and temperature suitable for bleaching. The termincludes, but is not limited to hydrogen peroxide and peracids.

As used herein, “oxidative stability” refers to the ability of a proteinto function under oxidative conditions. In particular, the term refersto the ability of a protein to function in the presence of variousconcentrations of H₂O₂ and/or peracid. Stability under various oxidativeconditions can be measured either by standard procedures known to thosein the art. A substantial change in oxidative stability is evidenced byat least about a 5% or greater increase or decrease (in mostembodiments, it is preferably an increase) in the half-life of theenzymatic activity, as compared to the enzymatic activity present in theabsence of oxidative compounds.

As used herein, the term “pH stability,” with respect to a protein,refers to the ability of a protein to function and/or remain active at aparticular pH. In general, most enzymes have a finite pH range at whichthey will function, and are stable. In addition to enzymes that functionin mid-range pHs (i.e., around pH 7), there are enzymes that are capableof working under conditions with very high or very low pHs. Stability atvarious pHs can be measured either by standard procedures known to thosein the art. A substantial change in pH stability is evidenced by atleast about 5% or greater increase or decrease (in most embodiments, itis preferably an increase) in the half-life of the enzymatic activity,as compared to the enzymatic activity at the enzyme's optimum pH.However, it is not intended that the present processes, methods and/orcompositions described herein be limited to any pH stability level norpH range.

As used herein, “thermal stability,” with respect to a protein, refersto the ability of a protein to function and/or remain active at aparticular temperature. In general, most enzymes have a finite range oftemperatures at which they will function and remain active. In additionto enzymes that work in mid-range temperatures (e.g., room temperature),there are enzymes that are capable of working in very high or very lowtemperatures. Thermal stability can be measured either by knownprocedures. A substantial change in thermal stability is evidenced by atleast about 5% or greater increase or decrease in the half-life of thecatalytic activity of a mutant when exposed to a different temperature(i.e., higher or lower) than optimum temperature for enzymatic activity.However, it is not intended that the processes, methods and/orcompositions described herein be limited to any temperature stabilitylevel nor temperature range.

As used herein, the term “chemical stability,” with respect to aprotein, refers to the stability of a protein (e.g., an enzyme) towardschemicals that adversely affect its activity. In some embodiments, suchchemicals include, but are not limited to hydrogen peroxide, peracids,anionic surfactants, cationic surfactants, non-ionic surfactants,chelants, and the like. However, it is not intended that the processes,methods and/or compositions described herein be limited to anyparticular chemical stability level nor range of chemical stability.

As used herein, the terms “purified” and “isolated” refer to the removalof contaminants from a sample and/or to a material (e.g., a protein,nucleic acid, cell, etc.) that is removed from at least one componentwith which it is naturally associated. For example, these terms mayrefer to a material which is substantially or essentially free fromcomponents which normally accompany it as found in its native state,such as, for example, an intact biological system

As used herein, the term “polynucleotide” refers to a polymeric form ofnucleotides of any length and any three-dimensional structure andsingle- or multi-stranded (e.g., single-stranded, double-stranded,triple-helical, and the like), which contain deoxyribonucleotides,ribonucleotides, and/or analogs or modified forms ofdeoxyribonucleotides or ribonucleotides, including modified nucleotidesor bases or their analogs. Because the genetic code is degenerate, morethan one codon may be used to encode a particular amino acid, and thepresent compositions and methods encompasses polynucleotides whichencode a particular amino acid sequence. Any type of modified nucleotideor nucleotide analog may be used, so long as the polynucleotide retainsthe desired functionality under conditions of use, includingmodifications that increase nuclease resistance (e.g., deoxy, 2′-O-Me,phosphorothioates, etc.). Labels may also be incorporated for purposesof detection or capture, for example, radioactive or nonradioactivelabels or anchors, e.g., biotin. The term polynucleotide also includespeptide nucleic acids (PNA). Polynucleotides may be naturally occurringor non-naturally occurring. The terms “polynucleotide” and “nucleicacid” and “oligonucleotide” are used interchangeably. Polynucleotidesmay contain RNA, DNA, or both, and/or modified forms and/or analogsthereof. A sequence of nucleotides may be interrupted by non-nucleotidecomponents. One or more phosphodiester linkages may be replaced byalternative linking groups. These alternative linking groups include,but are not limited to, embodiments wherein phosphate is replaced byP(O)S (“thioate”), P(S)S (“dithioate”), (O)NR₂ (“amidate”), P(O)R,P(O)OR′, CO or CH₂ (“formacetal”), in which each R or R′ isindependently H or substituted or unsubstituted alkyl (1-20 C)optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl,cycloalkenyl or araldyl. Not all linkages in a polynucleotide need beidentical. Polynucleotides may be linear or circular or comprise acombination of linear and circular portions.

As used herein, the term “polypeptide” refers to any compositioncomprised of amino acids and recognized as a protein by those of skillin the art. The conventional one-letter or three-letter codes for aminoacid residues are used. The terms “polypeptide” and “protein” are usedinterchangeably to refer to polymers of amino acids of any length. Thepolymer may be linear or branched, it may comprise modified amino acids,and it may be interrupted by non-amino acids. The terms also encompassan amino acid polymer that has been modified naturally or byintervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids, and the like), as well as other modifications known in theart.

As used herein, the term “related proteins” refers to functionallyand/or structurally similar proteins. In some embodiments, theseproteins are derived from a different genus and/or species, includingdifferences between classes of organisms (e.g., a bacterial protein anda fungal protein). In additional embodiments, related proteins areprovided from the same species. Indeed, it is not intended that theprocesses, methods and/or compositions described herein be limited torelated proteins from any particular source(s). In addition, the term“related proteins” encompasses tertiary structural homologs and primarysequence homologs. In further embodiments, the term encompasses proteinsthat are immunologically cross-reactive.

As used herein, the term “derivative” refers to a protein which isderived from a protein by addition of one or more amino acids to eitheror both the C- and N-terminal end(s), substitution of one or more aminoacids at one or a number of different sites in the amino acid sequence,and/or deletion of one or more amino acids at either or both ends of theprotein or at one or more sites in the amino acid sequence, and/orinsertion of one or more amino acids at one or more sites in the aminoacid sequence. The preparation of a protein derivative is preferablyachieved by modifying a DNA sequence which encodes for the nativeprotein, transformation of that DNA sequence into a suitable host, andexpression of the modified DNA sequence to form the derivative protein.

As used herein, the term “variant proteins” refers to related andderivative proteins. In some embodiments, a variant proteins differ froma parent (or parental) protein, e.g., a wild-type protein, by thepresence of different amino acid residues at a small number of aminoacid positions. The number of different amino acid residues may be oneor more, for example, 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, or moreamino acid residues. The number of different amino acids may be between1 and 10. Variant proteins may have defined level of sequence identityto a reference protein (for example, the parental protein), such as atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, oreven at least 99% amino acid sequence identity. Alternatively oradditionally, a variant protein may differ from a reference or parentalprotein in the number of prominent regions (i.e., domains, epitopes, orsimilar structural or functional portions). For example, in someembodiments, variant proteins have 1, 2, 3, 4, 5, or 10 correspondingprominent regions that differ from the parent protein. Methods known inthe art are suitable for generating variants of the enzymes describedherein, including but not limited to site-saturation mutagenesis,scanning mutagenesis, insertional mutagenesis, random mutagenesis,site-directed mutagenesis, and directed-evolution, as well as variousother recombinant and combinatorial approaches.

As used herein, the term “analogous sequence” refers to a sequencewithin a protein that provides similar function, tertiary structure,and/or conserved residues as a reference protein (e.g., a protein ofinterest having a desirable structure or function). For example, inepitope regions that contain an alpha-helix or a beta-sheet structure,the replacement amino acids in the analogous sequence preferablymaintain the same specific structure. The term also refers to nucleotidesequences, as well as amino acid sequences. In some embodiments,analogous sequences are developed such that the replacement amino acidsresult in a variant enzyme showing a similar or improved function. Insome embodiments, the tertiary structure and/or conserved residues ofthe amino acids in the protein of interest are located at or near thesegment or fragment of interest. Thus, where the segment or fragment ofinterest contains, for example, an alpha-helix or a beta-sheetstructure, the replacement amino acids preferably maintain that specificstructure.

As used herein, the term “homologous protein” refers to a protein (e.g.,perhydrolase) that has similar action and/or structure, as a referenceprotein (e.g., a protein of interest, such as a perhydrolase, fromanother source). It is not intended that homologs be necessarily relatedevolutionarily. Thus, it is intended that the term encompass the same orsimilar enzyme(s) (i.e., in terms of structure and function) obtainedfrom different species. In some embodiments, it is desirable to identifya homolog that has a quaternary, tertiary and/or primary structuresimilar to the protein of interest, as replacement for the segment orfragment in the protein of interest with an analogous segment from thehomolog will reduce the disruptiveness of the change. In someembodiments, homologous proteins induce similar immunologicalresponse(s) as a protein of interest. In some embodiments, homologousproteins are engineered to produce enzymes with desired activity(ies).

As used herein, the terms “wild-type” and “native,” with respect toproteins and nucleic acids, refer to those found in nature. The terms“wild-type sequence,” and “wild-type gene” are used interchangeablyherein, to refer to a sequence (protein or nucleic acid) that is nativeor naturally occurring in a host cell. In some embodiments, thewild-type sequence refers to a sequence of interest that is the startingpoint of a protein engineering project. The genes encoding thenaturally-occurring protein may be obtained in accord with the generalmethods known to those skilled in the art. The methods generallycomprise synthesizing labeled probes having putative sequences encodingregions of the protein of interest, preparing genomic libraries fromorganisms expressing the protein, and screening the libraries for thegene of interest by hybridization to the probes. Positively hybridizingclones are then mapped and sequenced.

The degree of homology between sequences may be determined using anysuitable method known in the art (see, e.g., Smith and Waterman (1981)Adv. Appl. Math. 2:482; Needleman and Wunsch (1970) J. Mol. Biol.48:443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444;programs such as GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package (Genetics Computer Group, Madison, Wis.); andDevereux et al. (1984) Nucleic Acids Res. 12:387-95).

For example, PILEUP is a useful program to determine sequence homologylevels. PILEUP creates a multiple sequence alignment from a group ofrelated sequences using progressive, pairwise alignments. It can alsoplot a tree showing the clustering relationships used to create thealignment. PILEUP uses a simplification of the progressive alignmentmethod of Feng and Doolittle, (Feng and Doolittle (1987) J. Mol. Evol.35:351-60). The method is similar to that described by Higgins and Sharp(Higgins and Sharp (1989) CABIOS 5:151-53). Useful PILEUP parametersincluding a default gap weight of 3.00, a default gap length weight of0.10, and weighted end gaps. Another example of a useful algorithm isthe BLAST algorithm, described by Altschul et al., (Altschul et al.(1990) J. Mol. Biol., 215:403-10; and Karlin et al. (1993) Proc. Natl.Acad. Sci. USA 90:5873-87). One particularly useful BLAST program is theWU-BLAST-2 program (Altschul et al. (1996) Meth. Enzymol. 266:460-80).Parameters “W,” “T,” and “X” determine the sensitivity and speed of thealignment. The BLAST program uses as defaults a wordlength (W) of 11,the BLOSUM62 scoring matrix (Henikoff and Henikoff (1989) Proc. Natl.Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10,M′5, N′-4, and a comparison of both strands.

As used herein, the phrases “substantially similar” and “substantiallyidentical,” in the context of at least two nucleic acids orpolypeptides, typically means that a polynucleotide or polypeptidecomprises a sequence that has at least about 40% identity, at leastabout 50% identity, at least about 60% identity, at least about 75%identity, at least about 80% identity, at least about 90% identity, atleast about 91% identity, at least about 92% identity, at least about93% identity, at least about 94% identity, at least about 95% identity,at least about 96% identity, at least about 97% identity, at least about98% identity, at least about 99% identity, compared to the reference(i.e., wild-type) sequence. Sequence identity may be determined usingknown programs such as BLAST, ALIGN, and CLUSTAL using standardparameters. (See, e.g., Altschul, et al. (1990) J. Mol. Biol.215:403-10; Henikoff et al. (1989) Proc. Natl. Acad. Sci. USA 89:10915;Karin et al. (1993) Proc. Natl. Acad. Sci USA 90:5873; and Higgins etal. (1988) Gene 73:237-44). Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation. Also, databases may be searched using FASTA (Pearson et al.(1988) Proc. Natl. Acad. Sci. USA 85:2444-48). One indication that twopolypeptides are substantially identical is that the first polypeptideis immunologically cross-reactive with the second polypeptide.Typically, polypeptides that differ by conservative amino acidsubstitutions are immunologically cross-reactive. Thus, a polypeptide issubstantially identical to a second polypeptide, for example, where thetwo peptides differ only by a conservative substitution. Anotherindication that two nucleic acid sequences are substantially identicalis that the two molecules hybridize to each other under stringentconditions (e.g., within a range of medium to high stringency).

As used herein, the terms “size” or “sizing” refer to compounds used inthe textile industry to improve weaving performance by increasing theabrasion resistance and strength of the yarn. Size is usually made of,for example, starch or starch-like compounds.

As used herein, the terms “desize” or “desizing,” refer to the processof eliminating size, generally starch, from textiles usually prior toapplying special finishes, dyes or bleaches.

As used herein, the term “desizing enzyme(s)” refers to enzymes that areused to enzymatically remove size. Exemplary enzymes are amylases,cellulases, and mannanases.

As used herein, the terms “perhydrolyzation,” “perhydrolyze,” or“perhydrolysis,” refer to a reaction wherein a peracid is generated fromester and hydrogen peroxide substrates. In one embodiment, theperhydrolyzation reaction is catalyzed with a perhydrolase, e.g., acyltransferase or aryl esterase, enzyme. In some embodiments, a peracid isproduced by perhydrolysis of an ester substrate of the formulaR₁C(═O)OR₂, where R₁ and R₂ are the same or different organic moieties,in the presence of hydrogen peroxide (H₂O₂). In one embodiment, —OR₂ is—OH. In one embodiment, —OR₂ is replaced by —NH₂. In some embodiments, aperacid is produced by perhydrolysis of a carboxylic acid or amidesubstrate.

As used herein, the term “peracid,” refers to a molecule derived from acarboxylic acid ester which has been reacted with hydrogen peroxide toform a highly reactive product that is able to transfer one of itsoxygen atoms. It is this ability to transfer oxygen atoms that enables aperacid, for example, peracetic acid, to function as a bleaching agent.

As used herein, the term “scouring,” refers to removing impurities, forexample, much of the non-cellulosic compounds (e.g., pectins, proteins,wax, motes, etc.) that are naturally found in cotton or other textiles.In addition to the natural non-cellulosic impurities, scouring canremove residual materials introduced by manufacturing processes, such asspinning, coning, or slashing lubricants. In some embodiments, bleachingmay be employed to remove impurities from textiles.

As used herein, the term “bioscouring enzyme(s)” refers to an enzyme(s)capable of removing at least a portion of the impurities found in cottonor other textiles.

As used herein, the term “motes” refers to unwanted impurities, such ascotton seed fragments, leaves, stems, and other plant parts, which clingto the fiber even after a mechanical ginning process.

As used herein, the term “greige” (pronounced gray) refers to textilesthat have not received any bleaching, dyeing, or finishing treatmentafter being produced. For example, any woven or knit fabric off the loomthat has not yet been finished (desized, scoured, and the like),bleached, or dyed, is termed a greige textile. The textiles used in theexamples, infra, are greige textiles.

As used herein, the term “dyeing,” refers to applying a color, e.g., totextiles, especially by soaking in a coloring solution.

As used herein, the term “non-cotton cellulosic” fiber, yarn, or fabricmeans fibers, yarns, or fabrics which are comprised primarily of acellulose based composition other than cotton. Examples of suchcompositions include linen, ramie, jute, flax, rayon, lyocell, celluloseacetate, bamboo and other similar compositions which are derived fromnon-cotton cellulosics.

As used herein, the term “pectate lyase” refers to a type of pectinase.Pectinases are a group of enzymes that cleave glycosidic linkages ofpectic substances mainly poly(1,4-alpha-D-galacturonide) and itsderivatives (see Sakai et al. (1993) Advances in Applied Microbiology39:213-294). Preferably, the pectinase catalyzes the random cleavage ofalpha-1,4-glycosidic linkages in pectic acid (also calledpolygalacturonic acid) by transelimination, such as enzymes in the classpolygalacturonate lyase (PGL; EC 4.2.2.2), also known aspoly(1,4-alpha-D-galacturonide) lyase or pectate lyase.

As used herein, the term “pectin” denotes pectate, polygalacturonic acidand pectin, which may be esterified to a higher or lower degree.

As used herein, the term “cutinase,” refers to as a plant, bacterial orfungal derived lipolytic enzyme used in textile processing. Cutinasesare capable of hydrolyzing the substrate, cutin. Cutinases can breakdown fatty acid esters and other oil-based compositions that need to beremoved during textile processing (e.g., the scouring). In someembodiments, the cutinases has significant plant cutin hydrolysisactivity. In particular embodiments, the cutinase has hydrolyticactivity on the biopolyester polymer cutin found on the leaves ofplants. Suitable cutinases may be isolated from many different plant,fungal and bacterial sources.

As used herein, the term “α-amylase” refers to an enzyme that cleavesthe α(1-4)glycosidic linkages of amylose to yield maltose molecules(disaccharides of α-glucose). Amylases are digestive enzymes found insaliva and are also produced by many plants. Amylases break downlong-chain carbohydrates (such as starch) into smaller units. An“oxidative stable” α-amylase is an α-amylase that is resistive todegradation by oxidative means, when compared to non-oxidative stableα-amylase, especially when compared to the non-oxidative stableα-amylase form which the oxidative stable α-amylase was derived.

As used herein, the term “protease” refers to a protein capable ofcatalyzing the cleavage of a peptide bond.

As used herein, a “catalase” refers to an enzyme that catalyzes thedecomposition of hydrogen peroxide to hydrogen and oxygen.

As used herein, the term “wicking” refers to the passage of liquidsalong or through a textile material or a textile element of a coatedfabric, or along interstices formed by a textile element and a coatingpolymer of a coated fabric. Wicking involves a spontaneous transport ofa liquid driven into a porous system by capillary forces.

As used herein, the phrase “degree of polymerization” refers to thenumber of repeating units in the individual macromolecules in a polymer.Degree of polymerization may be based on a mass (weight) or a numberaverage.

As used herein, the terms “fastness” or “color fastness” refer toability of a material to resist color change, i.e., to retain itsoriginal hue, especially without fading, running, or changing whenwetted, washed, cleaned, or stored under normal conditions when exposedto light, heat, or other influences.

As used herein, the terms “handle” or “hand” refer to the quality of atextile material, e.g., fabric or yarn, assessed by the reactionobtained from the sense of touch. It is concerned with the judgment of,for example, roughness, smoothness, harshness, pliability, thickness,and other tactile parameters.

As used herein, the term “pilling” refers to the entangling of textilefibers during washing, dry cleaning, testing, or in wear to form ballsor pills which protrude from the surface of a fabric, and which are ofsuch density that light will not pass through them, so that, e.g., theycast a shadow. Pilling that occurs during normal wear may be simulated,for example, on a laboratory-testing machine by controlled rubbingagainst an elastomeric pad having specifically selected mechanicalproperties. The degree of pilling may be evaluated against standards onan arbitrary scale ranging from 5 (indicating no pilling) to 1(indicating very severe pilling).

As used herein, the term “surfactant” refers to a substance that reducessurface tension of a liquid.

As used herein, the term “emulsifier” refers to a substance thatpromotes the suspension of one liquid in another.

As used herein, the term “sequestering agent” refers to a substancecapable of reacting with metallic ions by forming a water-solublecomplex in which the metal is held in a non-ionizable form.

As used herein, the terms “batch process,” “batchwise process,” or“discontinuous process” refer to the processing of textiles in lots orbatches in which the entirety of each batch is subjected to a process orone stage of a process at a time.

As used herein, the term “exhaust process” refers to a batch process inwhich pretreatment chemicals and/or an enzymatic pretreatmentcomposition and dye are added simultaneously or sequentially in a singletextile treatment bath.

As used herein, the term “liquor ratio” refers to the ratio of theweight of liquor (liquid) employed in a textile treatment process to theweight of the textile treated.

Enzymatic Textile Bleaching Compositions

One aspect of the compositions and methods provides enzymatic bleachingcompositions and methods for bleaching textiles using thesecompositions. Textiles include cellulose-containing textiles, e.g.,textiles made from cotton, flax, hemp, ramie, cellulose, acetate,lyocell, viscose rayon, bamboo, and various cellulosic blends, as wellas textiles made from polyamide, polyacrylic, wool, or blends thereof.In some embodiments, the textile comprises a blend with elastane. Theenzymatic bleaching compositions and methods are particularly useful forbleaching textiles containing fibers that are sensitive to high pH andtemperature conditions. The enzymatic bleaching compositions and methodsare particularly useful in batch, exhaust, or discontinuous processes.

The enzymatic bleaching compositions contain a perhydrolase enzyme, anester substrate for the perhydrolase enzyme suitable for production of aperacid upon catalytic reaction of the perhydrolase enzyme on thesubstrate in the presence of hydrogen peroxide, a hydrogen peroxidesource, a surfactant and/or an emulsifier, a peroxide stabilizer, asequestering agent, and a buffer which maintains a pH of about 6 toabout 8 during a textile bleaching process using the enzymatic bleachingcomposition. The enzymatic bleaching composition may optionally furthercontain a bioscouring agent or enzyme and/or a desizing agent or enzyme.

The enzymatic bleaching compositions, when used in a textilepretreatment process, advantageously produce bleached textiles thatexhibit increased dye uptake, decreased textile damage due to thebleaching process, and/or a bulkier softer handle when compared topretreatment with a chemical bleaching composition that does not containthe perhydrolase enzyme. In some embodiments, the enzymatic bleachingcompositions, when used in a textile pretreatment process, producetextiles with reduced pilling propensity.

Perhydrolase Enzyme

The enzymatic bleaching compositions include one or more perhydrolaseenzymes. In some embodiments, the perhydrolase enzyme isnaturally-occurring (i.e., a perhydrolase enzyme encoded by the genomeof a cell). In some embodiments, the perhydrolase enzyme comprises,consists of, or consists essentially of an amino acid sequence that isat least about 80%, at least about 85%, at least about 90%, at leastabout 91%, at least about 92%, at least about 93%, at least about 94%,at least about 95%, at least about 96%, at least about 97%, at leastabout 98%, at least about 99%, or even at least about 99.5% identical tothe amino acid sequence of a naturally-occurring perhydrolase enzyme.

In some embodiments, a perhydrolase enzyme is a naturally occurring M.smegmatis perhydrolase enzyme. In some embodiments, a perhydrolaseenzyme comprises, consists of, or consists essentially of the amino acidsequence set forth in SEQ ID NO: 1 or a variant or homologue thereof. Insome embodiments, a perhydrolase enzyme comprises, consists of, orconsists essentially of an amino acid sequence that is at least about80%, at least about 85%, at least about 90%, at least about 91%, atleast about 92%, at least about 93%, at least about 94%, at least about95%, at least about 96%, at least about 97%, at least about 98%, atleast about 99%, or even at least about 99.5% identical to the aminoacid sequence set forth in SEQ ID NO: 1.

The amino acid sequence of M. smegmatis perhydrolase (SEQ ID NO: 1) is:

MAKRILCFGDSLTWGWVPVEDGAPTERFAPDVRWTGVLAQQLGADFEVIEEGLSARTTNIDDPTDPRLNGASYLPSCLATHLPLDLVIIMLGTNDTKAYFRRTPLDIALGMSVLVTQVLTSAGGVGTTYPAPKVLVVSPPPLAPMPHPWFQLIFEGGEQKTTELARVYSALASFMKVPFFDAGSVISTDGVDGIHFTEAN NRDLGVALAEQVRSLL

The corresponding polynucleotide sequence encoding M. smegmatisperhydrolase (SEQ ID NO:2) is:

5′-ATGGCCAAGCGAATTCTGTGTTTCGGTGATTCCCTGACCTGGGGCTGGGTCCCCGTCGAAGACGGGGCACCCACCGAGCGGTTCGCCCCCGACGTGCGCTGGACCGGTGTGCTGGCCCAGCAGCTCGGAGCGGACTTCGAGGTGATCGAGGAGGGACTGAGCGCGCGCACCACCAACATCGACGACCCCACCGATCCGCGGCTCAACGGCGCGAGCTACCTGCCGTCGTGCCTCGCGACGCACCTGCCGCTCGACCTGGTGATCATCATGCTGGGCACCAACGACACCAAGGCCTACTTCCGGCGCACCCCGCTCGACATCGCGCTGGGCATGTCGGTGCTCGTCACGCAGGTGCTCACCAGCGCGGGCGGCGTCGGCACCACGTACCCGGCACCCAAGGTGCTGGTGGTCTCGCCGCCACCGCTGGCGCCCATGCCGCACCCCTGGTTCCAGTTGATCTTCGAGGGCGGCGAGCAGAAGACCACTGAGCTCGCCCGCGTGTACAGCGCGCTCGCGTCGTTCATGAAGGTGCCGTTCTTCGACGCGGGTTCGGTGATCAGCACCGACGGCGTCGACGGAATCCACTTCACCGAGGCCAACAATCGCGATCTCGGGGTGGCCCTCGCGGAACAGGTGCGGAGCCTGCT GTAA-3′

In some embodiments, the perhydrolase enzyme comprises one or moresubstitutions at one or more amino acid positions equivalent toposition(s) in the M. smegmatis perhydrolase amino acid sequence setforth in SEQ ID NO: 1. In some embodiments, the perhydrolase enzymecomprises any one or any combination of substitutions of amino acidsselected from M1, K3, R4, I5, L6, C7, D10, S11, L12, T13, W14, W16, G15,V17, P18, V19, D21, G22, A23, P24, T25, E26, R27, F28, A29, P30, D31,V32, R33, W34, T35, G36, L38, Q40, Q41, D45, L42, G43, A44, F46, E47,V48, I49, E50, E51, G52, L53, S54, A55, R56, T57, T58, N59, I60, D61,D62, P63, T64, D65, P66, R67, L68, N69, G70, A71, S72, Y73, S76, C77,L78, A79, T80, L82, P83, L84, D85, L86, V87, N94, D95, T96, K97,Y99F100, R101, R102, P104, L105, D106, I107, A108, L109, G110, M111,S112, V113, L114, V115, T116, Q117, V118, L119, T120, S121, A122, G124,V125, G126, T127, T128, Y129, P146, P148, W149, F150, I153, F154, I194,and F196.

In some embodiments, the perhydrolase enzyme comprises one or more ofthe following substitutions at one or more amino acid positionsequivalent to position(s) in the M. smegmatis perhydrolase amino acidsequence set forth in SEQ ID NO: 1: L12C, Q, or G; T25S, G, or P; L53H,Q, G, or S; S54V, L A, P, T, or R; A55G or T; R67T, Q, N, G, E, L, or F;K97R; V125S, G, R, A, or P; F154Y; F196G.

In some embodiments, the perhydrolase enzyme comprises a combination ofamino acid substitutions at amino acid positions equivalent to aminoacid positions in the M. smegmatis perhydrolase amino acid sequence setforth in SEQ ID NO: 1: L12I S54V; L12M S54T; L12T S54V; L12Q T25S S54V;L53H S54V; S54P V125R; S54V V125G; S54V F196G; S54V K97R V125G; or A55GR67T K97R V125G.

In some embodiments, the perhydrolase enzyme has aperhydrolysis:hydrolysis ratio of at least 1. In some embodiments, theperhydrolase enzyme has a perhydrolysis:hydrolysis ratio greater than 1.

In some embodiments, the perhydrolase enzyme is provided in theenzymatic bleaching composition at a concentration of about 1 to about2.5 pm, about 1.5 to about 2.0 ppm, or about 1.7 ppm.

Ester Substrate

The present enzymatic bleaching compositions further include an ester,which serves as a substrate for the perhydrolase enzyme for productionof a peracid in the presence of hydrogen peroxide. In some embodiments,the ester substrate is an ester of an aliphatic and/or aromaticcarboxylic acid or alcohol. In some embodiments, the ester substrate isan ester of one or more of the following: formic acid, acetic acid,propionic acid, butyric acid, valeric acid, caproic acid, caprylic acid,nonanoic acid, decanoic acid, dodecanoic acid, myristic acid, palmiticacid, stearic acid, and oleic acid. In some embodiments, triacetin,tributyrin, and other esters serve as acyl donors for peracid formation.In some embodiments, the ester substrate is propylene glycol diacetate,ethylene glycol diacetate, or ethyl acetate. In one embodiment, theester substrate is propylene glycol diacetate.

In some embodiments, the ester substrate is provided at a concentrationof about 2,000 to about 4,000 ppm, about 2,500 to about 3,500 ppm, about2,800 ppm to about 3,200 ppm, or about 3,000 ppm.

Hydrogen Peroxide Source

The present enzymatic bleaching compositions further include a hydrogenperoxide source. Hydrogen peroxide can be either added directly inbatch, or generated continuously “in situ” by chemical,electro-chemical, and/or enzymatic means.

In some embodiments, the hydrogen peroxide source is hydrogen peroxide.In some embodiments, the hydrogen peroxide source is a solid compoundthat generates hydrogen peroxide upon addition to water. Such compoundsinclude adducts of hydrogen peroxide with various inorganic or organiccompounds, of which the most widely employed is sodium carbonate perhydrate, also referred to as sodium percarbonate.

Inorganic perhydrate salts are one preferred embodiment of hydrogenperoxide source. Examples of inorganic perhydrate salts includeperborate, percarbonate, perphosphate, persulfate, and persilicatesalts. The inorganic perhydrate salts are normally the alkali metalsalts.

Other hydrogen peroxide adducts useful in the present compositionsinclude adducts of hydrogen peroxide with zeolites, or urea hydrogenperoxide.

The hydrogen peroxide source compounds may be included as a crystallineand/or substantially pure solid without additional protection. However,for certain granular perhydrate salts, the preferred forms are coatedwith a material that provides better storage stability. Suitablecoatings include inorganic salts such as alkali metal silicate,carbonate or borate salts or mixtures thereof, or organic materials suchas waxes, oils, or fatty soaps.

In some embodiments, the hydrogen peroxide source is an enzymatichydrogen peroxide generation system. In one embodiment, the enzymatichydrogen peroxide generation system comprises an oxidase and itssubstrate. Suitable oxidase enzymes include, but are not limited to:glucose oxidase, sorbitol oxidase, hexose oxidase, choline oxidase,alcohol oxidase, glycerol oxidase, cholesterol oxidase, pyranoseoxidase, carboxyalcohol oxidase, L-amino acid oxidase, glycine oxidase,pyruvate oxidase, glutamate oxidase, sarcosine oxidase, lysine oxidase,lactate oxidase, vanillyl oxidase, glycolate oxidase, galactose oxidase,uricase, oxalate oxidase, and xanthine oxidase.

The following equation provides an example of a coupled system forenzymatic production of hydrogen peroxide.

It is not intended that the present compositions and methods be limitedto any specific enzyme, as any enzyme that generates H₂O₂ with asuitable substrate may be used. For example, lactate oxidases fromLactobacillus species which are known to create H₂O₂ from lactic acidand oxygen may be used. One advantage of the enzymatic generation ofacid (e.g., gluconic acid in the above example) is that this reduces thepH of a basic solution to the pH range in which a peracid is mosteffective in bleaching (i.e., at or below the pKa). Other enzymes (e.g.,alcohol oxidase, ethylene glycol oxidase, glycerol oxidase, amino acidoxidase, and the like) that can generate hydrogen peroxide may also beused in combination with perhydrolase enzymes and ester substrates togenerate peracids.

Hydrogen peroxide may also be generated electrochemically, for exampleusing a fuel cell fed oxygen and hydrogen gas.

In some embodiments, the hydrogen peroxide source is hydrogen peroxideprovided at a concentration of about 1,000 to about 3,000 ppm, about1,500 to about 2,500 ppm, about 2,000 ppm to about 2,200 ppm, or about2,100 ppm.

Surfactants and Emulsifiers

The present enzymatic textile bleaching compositions may further includeone or more, i.e., at least one, surfactant(s) and/or emulsifier(s).Suitable surfactants include, without limitation, nonionic (see, e.g.,U.S. Pat. No. 4,565,647, which is herein incorporated by reference);anionic; cationic; and zwitterionic surfactants (see, e.g., U.S. Pat.No. 3,929,678). Anionic surfactants include, without limitation, linearalkylbenzenesulfonate, α-olefinsulfonate, alkyl sulfate (fatty alcoholsulfate), alcohol ethoxysulfate, secondary alkanesulfonate, α-sulfofatty acid methyl ester, alkyl- or alkenylsuccinic acid, and soap.Non-ionic surfactants include, without limitation, alcohol ethoxylate,nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide,ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide,polyhydroxy alkyl fatty acid amide, and N-acyl N-alkyl derivatives ofglucosamine (“glucamides”).

In some embodiments, the enzymatic bleaching composition contains anon-ionic surfactant. In one embodiment, the non-ionic surfactant is analcohol ethoxylate.

A surfactant may be present at a concentration of about 5% to about 40%,about 20% to about 30%, or about 5% to about 10% (w/w).

In some embodiments, the enzymatic bleaching composition containsethoxylated isotridecanol at a concentration of about 5% to about 30%,about 10% to about 25%, or about 15% to about 20% (w/w).

Peroxide Stabilizers

The present enzymatic bleaching compositions may further include aperoxide stabilizer. Examples of peroxide stabilizers include, but arenot limited to, sodium silicate, sodium carbonate, acrylic polymers,magnesium salts, and phosphonic acid. In one embodiment, the peroxidestabilizer is phosphonic acid.

The peroxide stabilizer may be present in an enzymatic bleachingcomposition at a concentration of about 1% to about 5%, about 1% toabout 10%, or about 2% to about 8% (w/w).

Sequestering Agents

The present enzymatic bleaching compositions may further include asequestering agent. Examples of sequestering agents include, but are notlimited to, amino carboxylates, amino phosphonates,polyfunctionally-substituted aromatic chelating agents,polyhydroxy-carboxylic acids, aminopolycarboxylic acids,polyphosphonates, and polyacrylic acids, and mixtures thereof.Particular amino carboxylates useful as sequestering agents includeethylenediaminetetracetates, N-hydroxyethylethylenediaminetriacetates,nitrilotriacetates, ethylenediamine tetraproprionates, andtriethylenetetraaminehexacetates.

Polyfunctionally-substituted aromatic sequestering agents are alsouseful in the present compositions (see, e.g., U.S. Pat. No. 3,812,044,issued May 21, 1974, to Connor et al.). Preferred compounds of this typein acid form are dihydroxydisulfobenzenes such as1,2-dihydroxy-3,5-disulfobenzenediethylenetriaminepentaacetates, andethanoldiglycines, alkali metal, ammonium, and substituted ammoniumsalts therein and mixtures thereof.

Amino phosphonates are also suitable for use as sequestering agents inthe present compositions, particularly when at least low levels of totalphosphorus are permitted.

A biodegradable sequestering agent suitable for use herein isethylenediamine disuccinate (“EDDS”), especially the [S,S] isomer asdescribed in U.S. Pat. No. 4,704,233 (issued Nov. 3, 1987 to Hartman andPerkins).

In one embodiment, the sequestering agent is polyacrylic acid.

A sequestering agent may be present in an enzymatic bleachingcomposition described herein at a concentration of about 1% to about15%, about 5% to about 10%, or about 3% to about 10% (w/w).

Buffers

The present enzymatic bleaching compositions may include a buffer thatis capable of maintaining the pH of the composition at a pH of about 6to about 8. In one embodiment, the buffer is a phosphate buffer, forexample, 100 mM phosphate buffer, pH 8.

Enzymatic Textile Bleaching Methods

Another aspect of the compositions and methods provides methods forbleaching of textiles, using any of the enzymatic bleaching compositionsdescribed herein. Generally, the textile to be bleached is contactedwith an enzymatic textile composition as described herein for a lengthof time and under conditions suitable to permit measurable whitening ofthe textile.

Textiles include cellulose-containing textiles, e.g., textiles made fromcotton, flax, hemp, ramie, cellulose, acetate, lyocell, viscose rayon,bamboo, and various cellulosic blends, as well as textiles made frompolyamide, polyacrylic, wool, or blends thereof. In some embodiments,the textile comprises a blend with elastane. The enzymatic bleachingcompositions and methods are particularly useful for bleaching textilescontaining fibers that are sensitive to high pH and temperatureconditions.

Advantageously, treatment of textiles in accordance with the methodsproduces bleached textiles with increased dye uptake, decreased textiledamage, and/or bulkier softer handle when compared to a chemicalbleaching process using a chemical bleaching composition that does notinclude a perhydrolase enzyme. In some embodiments, textiles areproduced that have a reduced pilling process when compared with achemical bleaching process that does not include a perhydrolase enzyme.

The present enzymatic bleaching further advantageously require lessenergy due to the lower processing temperatures that are employed incomparison to a typical chemical bleaching process. In addition, lessrinsing is required than a chemical bleaching process, resulting inlower water usage. The present methods also produces a lower pH effluent(<8) than chemical bleaching (about 13), resulting in reduced adverseenvironmental impact.

Typically, the present methods utilize a liquor ratio of about 6:1 toabout 15:1, for example, about 10:1. In some embodiments, the methodsare performed in a batch, exhaust, or discontinuous textile bleachingprocess.

Textiles are contacted with the enzymatic bleaching composition at atemperature of about 40° to about 70° C., for example, about 60° C. toabout 70° C., for a processing time about 40 to about 60 minutes. In oneembodiment, the bleaching temperature is about 65° C. and the processingtime is about 50 minutes. In some embodiments, the temperature of theenzymatic bleaching composition is raised by about 3° C. per minute froma starting temperature of about 20° C. to about 50° C. until theprocessing temperature for bleaching is reached.

In some embodiments, one or more rinsing steps are performed afterincubation of the textile in the enzymatic bleaching composition, toremove the bleaching composition. Typically, the textile is rinsed withan aqueous composition (water or a composition containing water). Insome embodiments, the rinsing temperature is about 40° C. to about 60°C., for example, about 50° C. In some embodiments, the aqueous rinsingcomposition contains a catalase enzyme to hydrolyze the hydrogenperoxide. In one embodiment, the textile is rinsed twice with a catalasecontaining aqueous composition for about 10 minutes for each rinse.

In some embodiments, textiles bleached using the methods herein containa softer, bulkier, and more natural handle than textiles bleachedpropensity when compared to a textile treated with a chemical bleachingcomposition that does not comprise a perhydrolase enzyme. This bulkier,softer handle often results in an improvement in sewability (needleresistance) and stretch. Further, the permanent bulkier, softer handleoften results in improvement in crease recovery, e.g., lower risk forcrease marking in piece good and garment processing.

In some embodiments, properties of elastane are enhanced using theenzymatic bleaching methods herein, in comparison to bleaching with achemical process that does not comprise a perhydrolase enzyme.

In some embodiments, the enzymatic bleaching methods herein result innatural fibers with less swelling and avoidance of channeling effect inyarn cheese dyeing machines, in comparison to a chemical bleachingprocess that does not comprise a perhydrolase enzyme.

Bioscouring Enzymes

In some embodiments, the present compositions and methods for enzymatictextile bleaching include one or more bioscouring enzymes. Thebioscouring enzyme(s) may be included in the enzymatic textile bleachingcomposition, or a textile may be treated with the bioscouring enzyme(s)in a subsequent processing step after pretreatment in the enzymatictextile bleaching composition. Exemplary bioscouring enzymes aredescribed, below.

Pectinases

Any pectinolytic enzyme having the ability to degrade the pectincomponent of, e.g., plant cell walls, may be used in the presentcompositions and methods. Suitable pectinases include, withoutlimitation, those of fungal or bacterial origin. The pectinases may beof natural origin or recombinantly produced, and/or may be chemically orgenetically modified. In some embodiments, the pectinases aremono-component enzymes.

Pectinases can be classified according to their preferential substrate,highly methyl-esterified pectin or low methyl-esterified pectin andpolygalacturonic acid (pectate), and their reaction mechanism,β-elimination or hydrolysis. Pectinases can be mainly endo-acting,cutting the polymer at random sites within the chain to give a mixtureof oligomers, or they may be exo-acting, attacking from one end of thepolymer and producing monomers or dimers. Several pectinase activitiesacting on the smooth regions of pectin are included in theclassification of enzymes provided by Enzyme Nomenclature (1992), e.g.,pectate lyase (EC 4.2.2.2), pectin lyase (EC 4.2.2.10),polygalacturonase (EC 3.2.1.15), exo-polygalacturonase (EC 3.2.1.67),exo-polygalacturonate-lyase (EC 4.2.2.9) andexo-poly-alpha-galacturonosidase (EC 3.2.1.82). In preferredembodiments, the methods utilize pectate lyases.

Pectate lyase enzymatic activity as used herein refers to catalysis ofthe random cleavage of α-1,4-glycosidic linkages in pectic acid (alsocalled polygalacturonic acid) by transelimination. Pectate lyases arealso termed polygalacturonate lyases and poly(1,4-D-galacturonide)lyases. For purposes of the present compositions and methods, pectatelyase enzymatic activity is the activity determined by measuring theincrease in absorbance at 235 nm of a 0.1% w/v solution of sodiumpolygalacturonate in 0.1 M glycine buffer at pH 10 (See Collmer et al.(1988) Methods Enzymol 161:329-35). Enzyme activity is typicallyexpressed as x mol/min, i.e., the amount of enzyme that catalyzes theformation of x mole product/min. An alternative assay measures thedecrease in viscosity of a 5% w/v solution of sodium polygalacturonatein 0.1 M glycine buffer at pH 10, as measured by vibration viscometry(APSU units). It will be understood that any pectate lyase may be usedin practicing the present compositions and methods.

Non-limiting examples of pectate lyases whose use is encompassed by thepresent present compositions and methods include pectate lyases thathave been cloned from different bacterial genera such as Erwinia,Pseudomonas, Bacillus, Klebsiella and Xanthomonas. Pectate lyasessuitable for use herein are from Bacillus subtilis (Nasser et al. (1993)FEBS Letts. 335:319-26) and Bacillus sp. YA-14 (Kim et al. (1994)Biosci. Biotech. Biochem. 58:947-49). Other pectate lyases produced byBacillus pumilus (Dave and Vaughn (1971) J. Bacteriol. 108:166-74), B.polymyxa (Nagel and Vaughn (1961) Arch. Biochem. Biophys. 93:344-52), B.stearothermophilus (Karbassi and Vaughn (1980) Can. J. Microbiol.26:377-84), Bacillus sp. (Hasegawa and Nagel (1966) J. Food Sci.31:838-45) and Bacillus sp. RK9 (Kelly and Fogarty (1978) Can. J.Microbiol. 24:1164-72) have also been described and are contemplated tobe used in the present compositions and methods. Any of the above, aswell as divalent cation-independent and/or thermostable pectate lyases,may be used in practicing the present compositions and methods. In someembodiments, the pectate lyase comprises, for example, those disclosedin WO 04/090099 (Diversa) or WO 03/095638 (Novozymes).

An effective amount of pectolytic enzyme to be used according to themethod of the present compositions and methods depends on many factors,but according to the present compositions and methods the concentrationof the pectolytic enzyme in the aqueous medium may be from about 0.0001%to about 1% μg enzyme protein by weight of the fabric, such as about0.0005% to about 0.2% enzyme protein by weight of the fabric, or about0.001% to about 0.05% enzyme protein by weight of the fabric.

Enzymes that Hydrolyze Polyester Substrates

Any enzyme that hydrolyzes a polyester substrate is suitable for use inthe present compositions and methods, for example, a cutinase or lipase,including, for example, the enzyme derived from Humicola insolens strainDSM 1800, as described in Example 2 of U.S. Pat. No. 4,810,414 or, inone embodiment, the enzyme from Pseudomonas mendocina described in U.S.Pat. No. 5,512,203, variants thereof and/or equivalents. Suitablevariants are described, for example, in WO 03/76580. These documents areincorporated herein by reference.

Suitable bacterial enzymes may be derived from a Pseudomonas orAcinetobacter species, preferably from P. stutzeri, P. alcaligenes, P.pseudoalcaligenes, P. aeruginosa or A. calcoaceticus, most preferablyfrom P. stutzeri strain Thai IV 17-1 (CBS 461.85), PG-1-3 (CBS 137.89),PG-1-4 (CBS 138.89), PG-II-11.1 (CBS 139.89) or PG-II-11.2 (CBS 140.89),P. aeruginosa PAO (ATCC 15692), P. alcaligenes DSM 50342, P.pseudoalcaligenes IN 11-5 (CBS 468.85), P. pseudoalcaligenes M-1 (CBS473.85) or A. calcoaceticus Gr V-39 (CBS 460.85). With respect to theuse of enzymes derived from plants, it is known that enzymes thathydrolyze polyester substrates exist in the pollen of many plants andsuch enzymes would be useful in the present processes, methods andcompositions. Enzymes that hydrolyze polyester substrates may also bederived a from fungus, such as, Absidia spp.; Acremonium spp.; Agaricusspp.; Anaeromyces spp.; Aspergillus spp., including A. auculeatus, A.awamori, A. flavus, A. foetidus, A. fumaricus, A. fumigatus, A.nidulans, A. niger, A. oryzae, A. terreus and A. versicolor;Aeurobasidium spp.; Cephalosporum spp.; Chaetomium spp.; Coprinus spp.;Dactyllum spp.; Fusarium spp., including F. conglomerans, F.decemcellulare, F. javanicum, F. lini, F. oxysporum and F. solani;Gliocladium spp.; Humicola spp., including H. insolens and H.lanuginosa; Mucor spp.; Neurospora spp., including N. crassa and N.sitophila; Neocallimastix spp.; Orpinomyces spp.; Penicillium spp;Phanerochaete spp.; Phlebia spp.; Piromyces spp.; Pseudomonas spp.;Rhizopus spp.; Schizophyllum spp.; Trametes spp.; Trichoderma spp.,including T. reesei, T. reesei (longibrachiatum) and T. viride; andZygorhynchus spp. Similarly, it is envisioned that an enzyme thathydrolyzes a polyester substrate may be found in bacteria such asBacillus spp.; Cellulomonas spp.; Clostridium spp.; Myceliophthora spp.;Pseudomonas spp., including P. mendocina and P. putida; Thermomonosporaspp.; Thermomyces spp., including T. lanuginose; Streptomyces spp.,including S. olivochromogenes; and in fiber degrading ruminal bacteriasuch as Fibrobacter succinogenes; and in yeast including Candida spp.,including C. Antarctica, C. rugosa, C. torresii; C. parapsllosis; C.sake; C. zeylanoides; Pichia minuta; Rhodotorula glutinis; R.mucilaginosa; and Sporobolomyces holsaticus.

In some embodiments, enzymes that hydrolyze polyester substrates, forexample, a cutinase and/or a lipase, are incorporated in the enzymaticbleaching composition in an amount from about 0.00001% to about 2% ofenzyme protein by weight of the fabric, such as in an amount from about0.0001% to about 1% of enzyme protein by weight of the fabric, or in anamount from 0.005% to 0.5% of enzyme protein by weight of the fabric,often in an amount from about 0.001% to about 0.5% of enzyme protein byweight of the fabric.

Cellulases

Cellulases may be added to the present compositions and methods, e.g.,to promote bioscouring. Cellulases are classified as a series of enzymefamilies encompassing endo- and exo-activities as well as cellobiosehydrolyzing capability. The cellulase may be derived from microorganismswhich are known to be capable of producing cellulolytic enzymes, suchas, e.g., species of Humicola, Thermomyces, Bacillus, Trichoderma,Fusarium, Myceliophthora, Phanerochaete, Irpex, Scytalidium,Schizophyllum, Penicillium, Aspergillus or Geotricum. Known speciescapable for producing celluloytic enzymes include Humicola insolens,Fusarium oxysporum or Trichoderma reesei. Non-limiting examples ofsuitable cellulases are disclosed in U.S. Pat. No. 4,435,307; Europeanpatent application No. 0 495 257; PCT Patent Application No. WO91/17244;and European Patent Application No. EP-A2-271 004, all of which areincorporated herein by reference.

Cellulases are also useful for biopolishing of the textile. Cotton andother natural fibers based on cellulose can be improved by enzymaticbiopolishing to produce a fabric with a smoother and glossierappearance. The treatment is used to remove “fuzz,” i.e., the tinystrands of fiber that protrude from the surface of yarn. A ball of fuzzis called a “pill” in the textile trade. After biopolishing, the fuzzand pilling are reduced. The other benefits of removing fuzz are asofter and smoother handle and superior color brightness.

In some embodiments of the present compositions and methods, thecellulase may be used at a concentration in the range from about 0.0001%to about 1% enzyme protein by weight of the fabric, such as about0.0001% to about 0.05% enzyme protein by weight of the fabric, or about0.0001 to about 0.01% enzyme protein by weight of the fabric.

In some embodiments, one or more cellulase enzyme is included in theenzymatic textile bleaching composition as described herein, and asystem for removing hydrogen peroxide, e.g., catalase, is added afterthe bleached and biopolished textile is produced.

In some embodiments, a method for combined bleaching and biopolishing ofa textile is provided, comprising (i) contacting the textile with anenzymatic bleaching composition as described herein and a biopolishingenzyme, e.g., a cellulase enzyme, for a length of time and underconditions suitable to permit measurable whitening of the textile andbiopolishing of the textile, wherein the bleached and biopolishedtextile comprises at least one of decreased textile damage, bulkiersofter handle, and increased dye uptake when compared to a chemicalbleaching method that comprises contacting the textile with a chemicaltextile bleaching composition that does not comprise a perhydrolaseenzyme; and (ii) hydrolyzing hydrogen peroxide with a system forremoving hydrogen peroxide, e.g., a catalase enzyme, after the bleachedand biopolished textile is produced.

Determination of cellulase activity (ECU) The cellulolytic activity maybe determined in endo-cellulase units (ECU) by measuring the ability ofthe enzyme to reduce the viscosity of a solution of carboxymethylcellulose (CMC), The ECU assay quantifies the amount of catalyticactivity present in the sample by measuring the ability of the sample toreduce the viscosity of a solution of carboxy-methylcellulose (CMC). Theassay is carried out in a vibration viscosimeter (e.g., MIVI 3000 fromSofraser, France) at 40° C.; pH 7.5; 0.1 M phosphate buffer; time 30minutes using a relative enzyme standard for reducing the viscosity ofthe CHIC substrate (Hercules 7 LED), enzyme concentration approx. 0.15ECU/ml. The arch standard is defined to 8,200 ECU/g. One ECU is amountof enzyme that reduces the viscosity to one half under these conditions.

Other Bioscouring Enzymes

The present compositions and methods are not limited to the use of theenzymes discussed above for bioscouring. Other enzymes may be usedeither alone or in combination with each other or with those listedabove. For example, proteases may be used in the present compositionsand methods. Suitable proteases include those of animal, vegetable ormicrobial origin, preferably of microbial origin. The protease may be aserine protease or a metalloprotease, preferably an alkaline microbialprotease or a trypsin-like protease. Examples of proteases includeaminopeptidases, including prolyl aminopeptidase (3.4.11.5), X-proaminopeptidase (3.4.11.9), bacterial leucyl aminopeptidase (3.4.11.10),thermophilic aminopeptidase (3.4.11.12), lysyl aminopeptidase(3.4.11.15), tryptophanyl aminopeptidase (3.4.11.17), and methionylaminopeptidase (3.4.11.18); serine endopeptidases, includingchymotrypsin (3.4.21.1), trypsin (3.4.21.4), cucumisin (3.4.21.25),brachyurin (3.4.21.32), cerevisin (3.4.21.48) and subtilisin(3.4.21.62); cysteine endopeptidases, including papain (3.4.22.2),ficain (3.4.22.3), chymopapain (3.4.22.6), asclepain (3.4.22.7),actimidain (3.4.22.14), caricain (3.4.22.30) and ananain (3.4.22.31);aspartic endopeptidases, including pepsin A (3.4.23.1), AspergillopepsinI (3.4.23.18), Penicillopepsin (3.4.23.20) and Saccharopepsin(3.4.23.25); and metalloendopeptidases, including Bacillolysin(3.4.24.28).

Non-limiting examples of subtilisins include subtilisin BPN′, subtilisinamylosacchariticus, subtilisin 168, subtilisin mesentericopeptidase,subtilisin Carlsberg, subtilisin DY, subtilisin 309, subtilisin 147,thermitase, aqualysin, Bacillus PB92 protease, proteinase K, proteaseTW7, and protease TW3.

Commercially available proteases include ALCALASE™, SAVINASE.™,PRIMASE.™, DURALASE.™, ESPERASE™, KANNASE™, and DURAZYM™ (Novo NordiskA/S), MAXATASE.™, MAXACAL.™, MAXAPEM™, PROPERASE™, Purafect™, PURAFECTOXP™, FN2.™ and FN3™ (Genencor Division, Danisco US Inc.).

Also useful in the present compositions and methods are proteasevariants, such as those disclosed in patents or published patentapplications EP 130,756 (Genentech), EP 214,435 (Henkel), WO 87/04461(Amgen), WO 87/05050 (Genex), EP 251,446 (Genencor), EP 260,105(Genencor), Thomas et al. (1985) Nature 318:375-76, Thomas et al. (1987)J. Mol. Biol. 193:803-13, Russel et al. (1987) Nature 328:496-500, WO88/08028 (Genex), WO 88/08033 (Amgen), WO 89/06279 (Novo Nordisk A/S),WO 91/00345 (Novo Nordisk A/S), EP 525 610 (Solvay) and WO 94/02618(Gist-Brocades N.V.), all of which are incorporated herein by reference.

The activity of proteases can be determined as described in “Methods ofEnzymatic Analysis,” third edition, 1984, Verlag Chemie, Weinheim, vol.5.

In other embodiments, it is contemplated that lipases are used for thebioscouring of textiles either alone or with other bioscouring enzymesof the present compositions and methods. Suitable lipases (also, termedcarboxylic ester hydrolases) include, without limitation, those ofbacterial or fungal origin, including triacylglycerol lipases (3.1.1.3)and Phospholipase A2 (3.1.1.4). Lipases include, without limitation,lipases from Humicola (synonym Thermomyces), such as from H. lanuginosa(T. lanuginosus) as described in patents or published patentapplications EP 258,068 and EP 305,216 or from H. insolens as describedin WO 96/13580; a Pseudomonas lipase, such as from P. alcaligenes or P.pseudoalcaligenes (EP 218,272), P. cepacia (EP 331,376), P. stutzeri (GB1,372,034), P. fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720and WO 96/27002), P. wisconsinensis (WO 96/12012); a Bacillus lipase,such as from B. subtilis (Dartois et al. (1993) Biochem. Biophys. Acta1131:253-360); B. stearothermophilus (JP 64/744992) or B. pumilus (WO91/16422), all references are herein incorporated by reference. Otherexamples are lipase variants such as those described in WO 92/05249, WO94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292, WO 95/30744,WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO 97/07202, allof which are incorporated herein by reference. Preferred commerciallyavailable lipase enzymes include LIPOLASE™ and LIPOLASE ULTRA™,LIPOZYME™, PALATASE™, NOVOZYM™ 435 and LECITASE™ (all available fromNovo Nordisk A/S). The activity of the lipase can be determined asdescribed in “Methods of Enzymatic Analysis”, Third Edition, 1984,Verlag Chemie, Weinhein, vol. 4.

It will be understood that any enzyme exhibiting bioscouring activitymay be used in practicing the present compositions and methods. That is,bioscouring enzymes derived from other organisms, or bioscouring enzymesderived from the enzymes listed above in which one or more amino acidshave been added, deleted, or substituted, including hybrid polypeptides,may be used, so long as the resulting polypeptides exhibit bioscouringactivity. Such variants can be created using conventional mutagenesisprocedures and identified using, e.g., high-throughput screeningtechniques such as the agar plate screening procedure. For example,pectate lyase activity may be measured by applying a test solution to 4mm holes punched out in agar plates (such as, for example, LB agar),containing 0.7% w/v sodium polygalacturonate (Sigma P 1879). The platesare then incubated for 6 h at a particular temperature (such as, e.g.,75° C.). The plates are then soaked in either (i) 1 M CaCl₂ for 0.5 h or(ii) 1% mixed alkyl trimethylammonium Br (MTAB, Sigma M-7635) for 1 h.Both of these procedures cause the precipitation of polygalacturonatewithin the agar. Pectate lyase activity can be detected by theappearance of clear zones within a background of precipitatedpolygalacturonate. Sensitivity of the assay is calibrated usingdilutions of a standard preparation of pectate lyase.

Desizing Enzymes

In some embodiments, the methods for enzymatic textile bleachingdescribed herein include one or more desizing enzyme. One or moredesizing enzyme may be included in the enzymatic textile bleachingcomposition, or a textile may be treated with desizing enzyme(s) in asubsequent processing step after pretreatment in the enzymatic textilebleaching composition.

Any suitable desizing enzyme may be used in the present compositions andmethgods. In some embodiments, the desizing enzyme is an amylolyticenzyme. Mannanases and glucoamylases may also be used. In someembodiments, the desizing enzyme is an α- or β-amylase and combinationsthereof.

Amylases

Alpha and beta amylases, which are appropriate in the context of thepresent compositions and methods, include those of bacterial or fungalorigin. Chemically or genetically modified mutants of such amylases arealso included in this connection. Preferred α-amylases include, forexample, α-amylases obtainable from Bacillus species. Useful amylasesinclude but are not limited to OPTISIZE 40™, OPTISIZE 160™, OPTISIZE HT260™, OPTISIZE HT 520™, OPTISIZE HT Plus™, OPTISIZE FLEX™ (all fromGenencor), DURAMYL™, TERMAMYL™, FUNGAMYL™ and BAN™ (all available fromNovozymes A/S, Bagsvaerd, Denmark). Other preferred amylolytic enzymesare CGTases (cyclodextrin glucanotransferases, EC 2.4.1.19), e.g., thoseobtained from species of Bacillus, Thermoanaerobactor orThermoanaero-bacterium.

The activity of OPTISIZE 40™ and OPTISIZE 160™ is expressed in RAU/g ofproduct. One RAU is the amount of enzyme which will convert 1 gram ofstarch into soluble sugars in one hour under standard conditions. Theactivity of OPTISIZE HT 260™, OPTISIZE HT 520™ and OPTISIZE HT Plus™ isexpressed in TTAU/g. One TTAU is the amount of enzyme that is needed tohydrolyze 100 mg of starch into soluble sugars per hour under standardconditions. The activity of OPTISIZE FLEX™ is determined in TSAU/g. OneTSAU is the amount of enzyme needed to convert 1 mg of starch intosoluble sugars in one minute under standard conditions.

Dosage of the amylase varies depending on the process type. Smallerdosages would require more time than larger dosages of the same enzyme.However, there isn't an upper limit on the amount of desizing amylaseother than what may be dictated by the physical characteristics of thesolution. Excess enzyme does not hurt the fabric; it allows for ashorter processing time. Based on the foregoing and the enzyme utilizedthe following minimum dosages for desizing are suggested:

Minimum dosage (per Typical Range (per liter Amylase Product liter ofdesizing liquor) of desizing liquor) OPTISIZE 40 ™ 1,000 RAU 2,000-70,000 RAU OPTISIZE 160 ™ 1,000 RAU  2,000-70,000 RAU OPTISIZE HT26 ™ 0 1,000 TTAU 3,000-100,000 TTAU OPTISIZE HT 520 ™ 1,000 TTAU3,000-100,000 TTAU OPTISIZE HT Plus ™ 1,000 TTAU 3,000-100,000 TTAUOPTISIZE FLEX ™ 5,000 TSAU 13,000-65,000 TSAU

The desizing enzymes may be derived from the enzymes listed above inwhich one or more amino acids have been added, deleted, or substituted,including hybrid polypeptides, so long as the resulting polypeptidesexhibit desizing activity. Such variants useful in practicing thepresent compositions and methods can be created using conventionalmutagenesis procedures and identified using, e.g., high-throughputscreening techniques such as the agar plate screening procedure.

The desizing enzyme is added to the aqueous solution (i.e., the treatingcomposition) in an amount effective to desize the textile materials.Typically, desizing enzymes, such as α-amylases, are incorporated intothe treating composition in amount from about 0.00001% to about 2% ofenzyme protein by weight of the fabric, preferably in an amount fromabout 0.0001% to about 1% of enzyme protein by weight of the fabric,more preferably in an amount from about 0.001% to about 0.5% of enzymeprotein by weight of the fabric, and even more preferably in an amountfrom about 0.01% to about 0.2% of enzyme protein by weight of thefabric.

Textiles

The present compositions and methods provide textiles, e.g., bleachedtextiles, produced according to any of the enzymatic bleaching methodsdescribed herein. Bleached textiles produced by incubation withenzymatic textile bleaching compositions as described herein exhibit atleast one of decreased textile damage, increased dye uptake, and bulkiersofter handle when compared to bleached textiles prepared with achemical bleaching composition that does not contain the perhydrolaseenzyme. The present compositions and methods also provides dyed textilesproduced from bleached textiles that have been produced according to theenzymatic bleaching methods herein.

In some embodiments, the bleached and/or bleached and dyed textile is acellulose-containing textile, including but not limited to cotton, flax,hemp, ramie, cellulose acetate, lyocell, viscose rayon, bamboo, andvarious cellulosic blends. In some embodiments, the bleached and/orbleached and dyed textile is a polyamide, polyacrylic, or wool textile,or a blend thereof.

Kits

The compositions and methods may be provided in the form of a kit ofparts (i.e., a kit). In one embodiment, the kit provides perhydrolaseenzyme, with instructions for use of the perhydrolase enzyme in anenzymatic textile bleaching composition and/or enzymatic textilebleaching method as described herein. Suitable packaging is provided. Asused herein, “packaging” refers to a solid matrix or materialcustomarily used in a system and capable of holding within fixed limitscomponents of a kit as described herein, e.g., perhydrolase enzyme.

Instructions may be provided in printed form or in the form of anelectronic medium such as a floppy disc, CD, or DVD, or in the form of awebsite address where such instructions may be obtained.

The following examples are intended to illustrate, but not limit, thepresent compositions and methods.

EXAMPLES Example 1 Enzymatic Bleach Pretreatment of 100% Cotton SingleJersey Material

A comparison between enzymatic and chemical bleaching processes wasperformed using cotton jersey textile material in a batch process in aMathis AG Lab Jet apparatus.

Bleaching Compositions

The compositions shown in Table 1 were used in experiments as describedbelow.

TABLE 1 Bleaching Compositions Bleaching Composition 1 2 3 4 Clarite ®ONE ml/l 1.5 1.5 1.5 1.5 Phosphate Buffer, ml/l 10 10 pH 8 PropyleneGlycol ml/l 3.0 3.0 Diacetate Pectinase ml/l 2.5 NaOH 100% g/l 1.5 1.5H₂O₂ 35% ml/l 6.0 4.0 6.0 6.0 Perhydrolase g/l 1.0 1.0

CLARITE® ONE contained the following components:

0.5% (w/w) phosphonic acid [[(phosphonomethyl)imino]bis[2,1-ethanediylnitrilobis(methylene)]]tetrakis-, sodium salt5-10% (w/w) alkylethoxylate15-20% (w/w) isotridecanol, ethoxylated<5% (w/w) polyacrylic acid, sodium salt

The phosphate buffer contained 10% soda ash.

The pectinase was a 10% solution of BIOPREP™ 3000L, available fromNovozymes.

The perhydrolase was the S54V variant of SEQ ID NO:1 at a stockconcentration of 1.7 g/l.

Pretreatment Process

About 120 g of fabric was incubated in each pretreatment compositionwith a liquor ratio of about 10:1. The MathisAG Laboratory Jet machineraised the temperature of the bath by 3° C. per minute from ambienttemperature to a target temperature of 65° C. The bath was then held at65° C. for 50 minutes.

Two rinses were performed for 10 minutes each at 50° C. A 25% solutionof CATALASE T100™, available from Genencor, was included in each rinse.Peroxide concentration before and after rinsing is shown in Table 2 foreach bleaching composition tested. Peroxide concentration was assessedusing indicator strips from Merck.

TABLE 2 Peroxide Concentration Before and After Rinsing with CatalaseBleaching Composition 1 2 3 4 Before ppm 25 25 20 15 After ppm 0 0 0 0

Rewetting

Rewetting was assessed for fabric treated with each bleachingcomposition described above using a modified wicking test. Deionizedwater was placed in a beaker, a strip of fabric was added to the beakerjust touching the water, and the time was then measured for the water totravel 1 cm. Better hydrophilicity is indicated by a low rewetting rate,expressed in cm/sec. The results are shown in Table 3.

TABLE 3 Rewetting Values Bleaching Composition 1 2 3 4 Hydrophilicity1.5 2.7 104 80 (cm/sec)

Whiteness

Whiteness was quantified using four different test methods. The resultsare shown in Table 4.

TABLE 4 Degree of Whiteness Bleaching Composition 1 2 3 4 Ganz 50 46 2524 ISO/Tappi 86.0 85.4 80.1 78.9 CIE 73 71 60 58 Berger 72 69 59 58

Fabric Damage Assessment

Degree of polymerization was assessed for fabric treated with eachbleaching composition described above. Degree of polymerization wasdetermined using the Swiss EWN Method (Swiss standard SNV 195 598). Thedamage factor (S) was determined according to the formula from O.Eisenhut, relating fiber damage to the change in degree ofpolymerization value before and after pretreatment.

The results are presented in Table 5. For comparison, the degree ofpolymerization for grey 100% cotton knitgood was 2380.

TABLE 5 Fabric Damage Assessment Bleaching Composition 1 2 3 4 Degree of2060 2110 2280 2230 Polymerization Damage Factor s: 0.17 s: 0.15 s: 0.05s: 0.08

Dyeing and Color Fastness

Fabric treated with the bleaching compositions described above was dyedwith NOVACRON® Rot FN 3G, 3% (w/w), for 90 min at 60° C. in a MathisAGLabomat device. Dye depth, hue deviation, and chroma deviation wereassessed.

The results of the colorimetric assessment are shown in Table 6.Colorimetric assessment was based on the colorimetry CIE-Lab (Munsell0with hue deviation indicating differences in shade (red-green andblue-yellow) and chroma deviation indicating differences in brilliancy.

TABLE 6 Colorimetric Assessment Bleaching Composition 1 2 3 4 RelativeDye 100 100   100   104   Depth Hue Deviation — −0.2 −0.4 −0.3 (traceredder) (trace redder) Chroma — −0.3 −0.1 −0.6 Deviation (trace duller)(slightly duller)

Fastness was assessed as rubbing fastness, wash fastness, waterfastness, and perspiration acid and alkaline fastness. Wet/dry rubbingfastness (crocking) was assessed according to test method ISO 105-X12.Wash fastness was assessed at 60° C. according to test method ISO105-006. Water fastness was assessed according to test method ISO105-E01. Acid/alkaline perspiration fastness was assessed according totest method ISO 105-E04. For all of these parameters, similar resultswere obtained for the chemical bleaching compositions (1 and 2) and theenzymatic bleaching compositions (3 and 4).

Handle

A bulkier, softer fabric handle was observed with fabric that waspretreated in the enzymatic bleaching compositions (3 and 4) incomparison to fabric pretreated in the chemical bleaching compositions(1 and 2), both before and after dyeing.

Although the foregoing present compositions and methods have beendescribed in some detail by way of illustration and examples forpurposes of clarity of understanding, it will be apparent to thoseskilled in the art that certain changes and modifications may bepracticed without departing from the spirit and scope of the invention.Therefore, the description should not be construed as limiting the scopeof the invention.

All publications, patents, and patent applications cited herein arehereby incorporated by reference in their entireties for all purposesand to the same extent as if each individual publication, patent, orpatent application were specifically and individually indicated to be soincorporated by reference.

1-20. (canceled)
 21. A method for bleaching a textile, comprising contacting the textile with an enzymatic textile bleaching composition comprising: (i) a perhydrolase enzyme; (ii) an ester substrate for said perhydrolase enzyme; (iii) a hydrogen peroxide source; (iv) a surfactant and/or an emulsifier; (v) a peroxide stabilizer; (vi) a sequestering agent; and (vii) a buffer that maintains a pH of about 6 to about 8; for a length of time and under conditions suitable to permit measurable whitening of the textile, thereby producing a bleached textile, wherein the bleached textile comprises at least one of decreased textile damage, bulkier softer handle, and increased dye uptake when compared to a chemical textile bleaching method that comprises contacting the textile with a chemical textile bleaching composition that does not comprise a perhydrolase enzyme.
 22. The method of claim 21, further comprising hydrolyzing hydrogen peroxide with a catalase enzyme after the bleached textile is produced.
 23. The method of claim 21, wherein the liquor ratio is about 10:1.
 24. The method of claim 21 performed in a batch or exhaust process.
 25. The method of claim 21, wherein the method provides any of at least about 10, 20, 30, 40, or 50% less weight loss than a chemical bleaching composition that does not comprise a perhydrolase enzyme.
 26. The method of claim 21, wherein the method provides a textile capable of increased dye uptake to produce a dyed textile with at least about any of at least about 5, 10, 15, 20, 25, or 30% increased dye depth when compared to a textile treated with a chemical bleaching composition that does not comprise a perhydrolase enzyme.
 27. The method of claim 21, wherein the method provides a textile that exhibits reduced pilling propensity when compared to a textile treated with a chemical bleaching composition that does not comprise a perhydrolase enzyme.
 28. The method of claim 21, wherein the textile is contacted with the enzymatic textile bleaching composition at a bleaching temperature of about 60° to about 70° C. for a processing time of about 40 to about 60 minutes.
 29. The method of claim 28, wherein the temperature of the enzymatic textile bleaching composition is raised by about 3° C. per minute from a starting temperature of about 20° to about 40° C. until the bleaching temperature is reached.
 30. The method of claim 28, wherein the bleaching temperature is about 65° C. and the processing time is about 50 minutes.
 31. The method of claim 21, wherein the bleached textile is rinsed with an aqueous composition at a rinsing temperature of about 40° C. to about 60° C. to remove said enzymatic textile bleaching composition.
 32. The method of claim 31, wherein the rinsing temperature is about 50° C.
 33. The method of claim 31, wherein the rinsing comprises rinsing said bleached textile twice for about 10 minutes for each rinse.
 34. The method of claim 21, wherein the aqueous composition comprises a catalase enzyme to hydrolyze the hydrogen peroxide.
 35. (canceled) 